Preliminary Investigation Of Renalase And The Progression Of Lupus Nephritis

Objective Lupus nephritis remains one of the most common and severe manifestations of systemic lupus erythematosus associated with considerable morbidity and mortality.Currently,early diagnosis and effective intervention is still main measure to protect LN patients from progressing to end stage renal disease.There were some side effects of currently used medicines in LN treatment.Once patients got severe infections,the mortality rate increased.Therefore,it's essential to investigate new medicines with fewer side effects for the treatment of LN.Renalase is a flavinprotein that oxidizes catecholamines.Renalase and associated peptide RP220 also could attenuate ischemic kidney injury and associated with autoimmune diseases.Previous researchs showed that renalase and RP220 had the role of anti-inflammatory and were associated with inflammatory reactions.So we proposed that renalase and RP220 might participate in the progression of LN.This preliminary research is aimed to find 1)the role and mechniasm of RP220 in the progression of LN;2)the correlation between serum renalase with clinical,pathological parameters in LN;3)the relationship between serum renalase with disease activity in LN.Methods 1.1)12-week old MRL/lpr mice were randomly divided into veh group,RP220 group and CTX group.Urine albumin creatinine ratio was tested every week.Renal function and renal pathological changes were evaluated.The mice macrophages(RAW264.7)cultured in vitro were randomly divided into con group,RP220 group,LPS group,siRNA group,mock group,LPS+RP220 group,siRNA+ LPS group and mock+LPS group.Pro-inflammatory cytokines and chemokines were assessed by western blotting and real-time PCR.The expression of NLRP3 inflammasome and MAPK signaling pathways were detected by western blotting assay.2.Patients who underwent biopsy and diagnosed as LN in our center were enrolled.Healthy controls who assigned informed consent were also recruited.The clinical and pathological parameters were recalled.Serum renalase levels were determined by ELISA.Immunofluorescent was used to detect the renalase expression in renal of LN patients and helathy control.We compared serum renalase in LN patients with helathy control.The relationship between serum renalase with pathological classification and manifestations in patients with LN were also analysed.3.For the cross-sectional study,proliferative LN patients were enrolled.The association of serum renalase with disease activity in LN was assessed.For the prospective study,seventeen active proliferative LN patients were recruited and followed up for six months.The influence of treatment and remission on serum renalase levels was assessed.Results 1.1)RP220 could ameliorate lupus-like disease in MRL/lpr mice.Compared with veh groups,mice in RP220 group had lower blood urea nitrogen,urine albumin creatinine ratio and serum anti-dsDNA antibody concentrations.RP220 significantly ameliorated renal injury in MRL/lpr mice,attenuated mesangial cell proliferation and tubular injury.Mice in RP220 groups showed amelioration of arthrocele and hypertrophy of submaxillary gland compared with the veh group.2)The renal expression of pro-inflammatory cytokines TNF-?,IL-6,IL-1? and MCP-1 was significantly decreased in RP220 group,as well as the protein exprsssion of NLRP3.The expression of pro-inflammatory factors IL-1? and IL-6 in macrophages was significantly upregulated after LPS stimulation.Pretreatment macrophages with RP220 inhibited the synthesis of pro-inflammatory cytokines.Transfection of macrophages with siRNA to knock down renalase expression further aggravated LPS-induced pro-inflammatory cytokine synthesis.Western blotting results showed that MAPK signaling pathway in macrophages was activated after stimulated with LPS.RP220 had no significant effect on MAPK signaling pathway.2.Seventy patients with lupus nephritis(LN)and thirty-five healthy volunteers were enrolled.In patients with LN,three were male.The average age of LN patients was37.93±13.59 years old.Healthy volunteers have the comparable age and gender with LN paitents.Serum renalase levels were significantly higher in LN patients than that in healthy controls.Moreover,patients with proliferative LN had elevated serum renalase levels than those with pure Class V LN patients.The performance of renalase as a marker for diagnosis of proliferative LN was 0.78.When the cutoff value was set at 54.81?g/ml,the sensitivity for proliferative LN was 67.80% and the specificity was 85.71%.Serum renalase levels were found positivily correlated with activity index,cellular crescent and interstitial fibrosis.The renal expression of renalase was showed to be elevated in LN patients compared to healthy control.3.In the cross-sectional study,59 proliferative patients with LN were recruited.Thirty-two patients were divided into disease active group according to their SLEDAI scores,the other twenty-seven patients were in the inactive group.Serum renalase levels were significantly higher in patients with active LN compared to inactive LN.Univariate analysis of the data revealed positive correlations between serum renalase levels and 24 hr urine protein excretion,ESR,anti-dsDNA antibody,SLEDAI scores and r SLEDAI scores.The amount of serum renalase was negatively correlated with albumin and C3 levels.The area under the ROC curve of serum renalase as an indicator for active LN was 0.906,the sensitivity and specificity were 87.5% and 89.2%,respectively.In the prospective study,17 active LN patients were enrolled.Serum renalase levels were found to be significantly decreased after immunosuppressive treatment.Conclusions RP220 could attenuate disease activity and renal injury in MRL/lpr mice by inhibiting inflmmatory reactions.RP220 might be one of the new agents for LN treatment.Serum renalase levels were significantly higher in LN patients compared to healthy controls.Serum renalase was associated with renal pathological classification and disease activity in LN patients making it a potential marker for LN disease acitivity in future clinical studies.